The long-term goal of these studies is to understand the fundamental mechanisms that regulate the concentration of copper in human cells. Copper plays an essential role in human physiology and is required for embryonic development, respiration, neurotransmitter biosynthesis, and other important physiological processes. Human copper-transporting ATPases ATP7A and ATP7B play a central role in the regulation of intracellular copper. Mutations in these transporters result in the severe metabolic disorders, Menkes disease and Wilson's disease, respectively. Recent studies also suggest that ATP7A (Menkes disease protein, MNKP) and ATP7B (Wilson's disease protein, WNDP) are involved in cellular resistance to platinum- based chemotherapeutic drugs, and therefore could be important pharmacological targets in cancer treatment. Despite the essential role of MNKP and WNDP in human physiology, our knowledge of how these ATP-driven transporters regulate intracellular copper is extremely limited. In the proposed series of experiments, a unique set of biochemical tools established by the Pi's laboratory is utilized to dissect specific molecular mechanisms that control the function and regulation of WNDP and MNKP in cells. Four specific aims are proposed. The first objective is to generate a comprehensive picture of copper-dependent regulation of WNDP and MNKP by characterizing the functional roles of the multiple metal-binding sites in the N-terminal domain of these proteins. The second goal is to obtain structural information necessary for an understanding of nucleotide binding by WNDP and MNKP and for the development of specific inhibitors for these transporters. Thirdly, the role of a recently discovered protein Murrl in the function and regulation of WNDP will be determined using a combination of biochemical and cell biological approaches. The final objective is to dissect the molecular mechanism of the MNKP/WNDP-related cisplatin resistance. These studies will yield important mechanistic information about the regulation of copper in human cells and will contribute to a better understanding and treatment of disorders associated with copper misbalance.